Project Summary Tick-borne diseases are on the increase, and are responsible for nearly all of the vector-transmitted disease in the US. Vector-borne pathogens face the dual challenge of adaptation to two very different host environments: the arthropod vector and the mammalian host. To survive within eukaryotic cells, the rickettsial pathogen Anaplasma phagocytophilum blocks phago-lysosome maturation, inhibits apoptosis, modulates host gene expression, redirects trans-Golgi trafficking, and repurposes autophagic machinery to build a replication vacuole. A. phagocytophilum must accomplish this, all while evading the unique innate immune defenses of mammalian and arthropod host cells. All pathogens in the order Rickettsiales utilize a specialized Type IV Secretion System (T4SS) to deliver effector molecules into the host cell cytosol to mediate host pathogen interactions. However, identification of the secreted effectors has been limited by the obligate nature of these pathogens. Even less is known about how effectors contribute to rickettsial growth in tick cells, as the tick vector remains an understudied niche of these pathogens. To overcome this, our group has developed a T4SS effector prediction program Optimal-features Predictor for T4SS Effectors (OPT4e). When applied to A. phagocytophilum, OPT4e identified 48 putative T4SS effectors. Transcriptomics finds that 15 of these predicted effector genes are specifically expressed during growth within either tick or mammalian cells. We have demonstrated that one of these tick- specific effector candidates, Aph1383, is translocated in a T4 specific manner by the Legionella pneumophila T4SS. Aph1383 also belongs to a paralogous family of six proteins encoded by the gene cluster aph1380-1386. This entire cluster is expressed 2.5-fold more highly during A. phagocytophilum growth in tick cells than during mammalian cell infections. We hypothesize that this family of Aph1383 paralogs are all T4SS effectors which target host cell processes specifically important for A. phagocytophilum growth within tick cells. In this study, we will first use transposon insertion mutants in the aph1380-1386 gene cluster to test the fitness contribution of these genes during A. phagocytophilum growth within tick cells. Next, we will evaluate the T4SS translocation of all Aph1383 paralogs and identify the amino acid sequences necessary for secretion. Finally, we will identify the subcellular localization and molecular targets of Aph1383 within tick cells. Characterizing these molecular interactions of A. phagocytophilum within the tick cell will open the door to development of vector targeted interventions to reduce transmissibility of the pathogen.